agroecological areas in Senegal and Gambia (West Africa). A polyphasic approach including both phenotypic and genotypic techniques was used to study the ...
International Journal of Systematic and Evolutionary Microbiology (2000), 50, 159–170
Printed in Great Britain
Polyphasic characterization of rhizobia that nodulate Phaseolus vulgaris in West Africa (Senegal and Gambia) Adama Diouf,1 Philippe de Lajudie,2 Marc Neyra,1 Karel Kersters,3 Monique Gillis,3 Esperanza Martinez-Romero4 and Mamadou Gueye1 Author for correspondence : Mamadou Gueye. Tel : 221 849 33 21. Fax : 221 832 16 75. e-mail : mamadou.gueye!orstom.sn
1
MIRCEN/Centre ISRA-IRD, BP 1386, Dakar, Senegal, West Africa
2
Laboratoire des Symbioses Tropicales et Me! diterrane! ennes, IRD, Campus de Baillarguet, BP 5035, 34032 Montpellier Ce! dex 1, France
3
Laboratorium voor Microbiologie, Universiteit Gent, K.-L. Ledeganckstraat 35, B-9000 Ghent, Belgium
4
Centro de Investigacio! n sobre Fijacio! n de Nitro! geno, Universidad Nacional Auto! noma de Me! xico, AP 565-A Cuernavaca, Morelos, Mexico
Fifty-eight new isolates were obtained from root nodules of common bean (Phaseolus vulgaris) cultivated in soils originating from different agroecological areas in Senegal and Gambia (West Africa). A polyphasic approach including both phenotypic and genotypic techniques was used to study the diversity of the 58 Rhizobium isolates and to determine their taxonomic relationships with reference strains. All the techniques performed, analysis of multilocus enzyme electrophoretic patterns, SDS-PAGE profiles of total cell proteins, PCR-RFLP analysis of the genes encoding 16S rRNA and of the 16S–23S RNA intergenic spacer region (ITS-PCR-RFLP), auxanographic tests using API galleries and nodulation tests lead to the consensus conclusion that the new rhizobial isolates formed two main distinct groups, I and II, belonging to Rhizobium tropici type B and Rhizobium etli, respectively. By MLEE R. etli and group II strains showed several related electrophoretic types, evidencing some extent of internal heterogeneity among them. This heterogeneity was confirmed by other techniques (ITS-PCR-RFLP, SDS-PAGE and host-plantspecificity) with the same nine distinct strains of group II showing some differences from the core of group II (54 strains).
Keywords : Rhizobium, root nodules, common bean (Phaseolus vulgaris)
INTRODUCTION
During recent years the classification of rhizobia that nodulate common bean (Phaseolus vulgaris) plants has been progressively revised as more rhizobial diversity is gradually discovered in different parts of the world (Martı! nez-Romero et al., 1991 ; Segovia et al., 1993 ; Amarger et al., 1997). These rhizobia were initially assigned to Rhizobium leguminosarum bv. phaseoli on the basis of their host specificity, separate from R. leguminosarum bv. viciae and R. leguminosarum bv. trifolii, symbionts of peas (Pisum spp., Vicia spp.) and clovers (Trifolium spp.) respectively (Jordan, 1984). This subdivision was also supported by their different symbiotic plasmids, encoding the nodulation specificities (Martı! nez et al., 1985, 1988). R. leguminosarum bv. phaseoli was long recognized to be taxonomically heterogeneous as evidenced by protein profile analysis .................................................................................................................................................
Abbreviations : ET, electrophoretic type ; ITS, internal transcribed spacer ; MLEE, multilocus enzyme electrophoresis. 01041 # 2000 IUMS
(Roberts et al., 1980), multilocus enzyme electrophoresis (MLEE) (Pin4 ero et al., 1988 ; Eardly et al., 1995), DNA relatedness analysis (Laguerre et al., 1993 ; van Berkum et al., 1996) and 16S rRNA genes (rDNA) sequencing (Segovia et al., 1993 ; Laguerre et al., 1993 ; Hernandez-Lucas et al., 1995). R. leguminosarum bv. phaseoli from Mexico and South America was first divided into R. leguminosarum bv. phaseoli type I and type II (Martı! nez et al., 1988). Rhizobium tropici types A and B were first proposed for type II strains carrying a single nifH gene copy. R. tropici types A and B are distinguished by their DNA–DNA hybridization values, a number of phenotypic characteristics and the presence of a specific megaplasmid (Martı! nez-Romero et al., 1991 ; Geniaux et al., 1995). Rhizobium etli was then proposed for R. leguminosarum bv. phaseoli type I strains which contain multiple copies of the nitrogenase reductase gene (nifH) on their symbiotic plasmids (Martı! nez et al., 1985 ; Segovia et al., 1993). More recently, two additional groups of Rhizobium strains nodulating bean plants were characterized in European soils and proposed as two new species, 159
A. Diouf and others
Rhizobium gallicum and Rhizobium giardinii (Amarger et al., 1997). Phaseolus vulgaris is commonly reported to originate from America whereas its symbiotic rhizobial partners are thought to be diverse and widely spread around the world. Native rhizobia nodulating common bean in African soils have been reported to be taxonomically related to R. tropici in East and South Africa (Anyango et al., 1995 ; Dagutat & Steyn, 1995) and to R. tropici and R. etli in Central Africa (Tjahjoleksono, 1993). In this paper, we studied the diversity and taxonomic relationships among 58 rhizobial strains originating from different agroecological areas in Senegal and Gambia. Using techniques with various discriminative powers such as physiological and auxanographic tests, host specificity, MLEE, analysis of total cell protein profiles by SDS-PAGE, RFLP analysis of PCRamplified ITS (internal transcribed spacer) 16S–23S and 16S rDNA we showed that, in West Africa, Phaseolus vulgaris is naturally nodulated by Rhizobium strains belonging to R. tropici type B and R. etli. METHODS Bacterial strains. Rhizobial strains (Table 1) were isolated
from root nodules of common bean cultivated during 15–20 d either in field conditions or in pots containing soils sampled at 5–20 cm depth in different places in Senegal and Gambia. These rhizobial strains were compared to several reference strains representing the different Rhizobium, Sinorhizobium, Mesorhizobium and Agrobacterium species or groups. Growth and culture conditions. New isolates and reference Rhizobium, Sinorhizobium and Mesorhizobium strains were maintained on yeast mannitol agar (YMA), containing (g l−") : mannitol, 10 ; sodium glutamate, 0±5 ; K HPO , 0±5 ; # , 0±004 % MgSO \7H O, 0±2 ; NaCl, 0±05 ; CaCl , 0±04 ; FeCl ; % # 1 ; pH 6±8 ; agar, 20. Agrobacterium # $ strains yeast extract, were maintained on yeast extract peptone-glucose medium, which contained (g l−" of 0±01 M phosphate buffer, pH 7±2) : peptone, 5 ; yeast extract, 1 ; beef extract, 5 ; sucrose, 5 and MgSO \7H O, 0±592. % # Morphological and physiological tests. Cell dimensions and morphology were determined by phase-contrast microscopy. Growth of strains was also studied on Luria broth (LB) medium containing (g l−") : bacto-tryptone, 50 ; yeast extract, 5 ; sodium chloride, 5 and on peptone yeast extract (PY) medium containing (g l−") : peptone, 5 ; yeast extract, 3 ; supplemented with 10 ml of sterile solution of CaCl 0±7 M. # was The maximum growth temperature on YM medium determined for all strains. PCR/RFLP of 16S–23S rDNA ITS region. Bacterial genomic DNA was extracted and purified as described by Boucher et al. (1987). Primers FGPS 1490, corresponding to positions 1521–1541 of Escherichia coli (Navarro et al., 1992), and FGPL 132«, corresponding to positions 114–132 of E. coli (Ponsonnet & Nesme, 1994), were used for PCR amplification. PCR was carried out in a 100 µl reaction volume by mixing 2 µl DNA extract with the polymerase reaction buffer (10 mM Tris}HCl, pH 8±3 ; 50 mM KCl, 0±0.1 % gelatin, 2 mM MgCl ) ; Taq polymerase (Bioprobe), 0±8 µl (5 # of the 10 µM dNTPs (dATP, dCTP, U}reaction) ; 2 µl each
160
dGTP, dTTP), 1 µl each primer (0±05 µM). PCR amplification was performed in a Gene Amp PCR System 2400 thermal cycler adjusted to the following temperature profile : initial denaturation at 94 °C for 5 min ; 35 amplification cycles (denaturation at 94 °C for 1 min, hybridization of primers at 55 °C for 1 min and extension at 72 °C for 1±5 min) ; final extension at 72 °C for 5 min. Amplification was checked by horizontal agarose (1 %, w}v) gel electrophoresis using 10 µl of the PCR product. Aliquots (8 µl) of PCR products were digested with the following restriction endonucleases (5 U}reaction) : AluI, DdeI, HhaI, PalI (Pharmacia), HinfI, MspI (Gibco-BRL), RsaI and TaqI (Boehringer Mannheim). Restricted DNA was analysed by horizontal agarose (NuSieve 3 : 1, FMC 3 %, w}v) gel electrophoresis. Electrophoresis was carried out at 60 V for 4 h and gels were stained as described above and photographed under UV illumination with Polaroid 665 positive}negative film. MLEE. Isolates and reference strains were grown overnight on an orbital shaker incubator at 30 °C in 30 ml PY medium. Cells were harvested by centrifugation at 6000 g for 10 min at 4 °C. After suspension in 0±2 ml MgSO 10 mM, the % bacteria were lysed by adding 40 µl of a lysozyme solution − (10 mg ml " MgSO 10 mM). The lysate maintained on ice was then placed for%15 min at room temperature and stored at ®70 °C for 10 min before use. Techniques of starch gel electrophoresis and selective staining of enzymes were performed as described by Selander et al. (1986). The electrophoretic buffer system, Tris-citrate (pH 8±0) was used for the following enzymes assayed : malate dehydrogenase, isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, xanthine dehydrogenase, phosphoglucomutase, esterases, aconitase, indophenol oxidase (super oxide dismutase), hexokinase and alanine dehydrogenase. Distinctive mobility variants (electromorphs) of each enzyme, numbered in order of decreasing anodal mobility, were equated with alleles at the corresponding gene locus, and electromorph profiles for the 12 enzymes (electrophoretic types, ETs) were considered to be multilocus genotypes (Table 2). The genetic diversity (h) for an enzyme locus was calculated from allele frequencies for ETs as h ¯ 1®Σxi# (n}(n®1)) where xi is the frequency of the ith allele and n is the number of ETs. The mean genetic diversity per locus (H) is the arithmetic mean of h values for the 12 loci (Selander et al., 1986). The genetic distance between each pair of ETs was estimated ; clustering at a matrix of pairwise genetic distances was performed by the unweighted pair group method with averages of Nei & Li (1979). PAGE of total bacterial proteins (SDS-PAGE). All strains were grown at 28 °C for 48 h in Roux flasks on tryptone yeast extract (TY) medium containing (g l−") : tryptone extract, 5 ; yeast extract, 0±75 ; KH PO , 0±454 ; Na HPO \12H O, # (Lab % M), 20 ; pH# 6±8–7. % Whole# 2±388 ; CaCl \6H O, 1 ; agar # cell protein #extracts were prepared from 80 mg cells and SDS-PAGE was performed using slight modifications of the Laemmli (1970) procedure, as described previously (Kiredjian et al., 1986). The normalized densitometric traces of the protein electrophoretic patterns were grouped by numerical analysis, using the GelCompar software package (Vauterin & Vauterin, 1992). The similarity between all pairs of traces was expressed by the Pearson product–moment correlation coefficient (r) converted for convenience to a percentage value (Pot et al., 1989, 1994). International Journal of Systematic and Evolutionary Microbiology 50
Phaseolus vulgaris-nodulating rhizobia Table 1. Rhizobium strains ISRA isolated from Phaseolus vulgaris and reference strains used in this study .................................................................................................................................................................................................................................................................................................................
Abbreviations : CIAT, Rhizobium Collection, Centro International de Agricultura Tropical, Cali, Columbia ; CIFN, Centro de Investigacio! n sobre Fijacio! n de Nitro! geno, Universidad Nacional Auto! noma de Me! xico, AP 565-A Cuernavaca, Morelos, Mexico ; ISRA, Institut Se! ne! galais de la Recherche Agricole, Centre ISRA-ORSTOM, BP 1386, Dakar, Senegal ; BCCM}LMGTM, Bacteria Collection, Laboratorium voor Microbiologie, K.-L. Ledeganckstraat 35, B9000 Gent, Belgium ; ORSTOM, Institut Français de Recherche Scientifique pour le De! veloppement en Coope! ration, BP 1386, Dakar, Senegal ; USDA, US Department of Agriculture, Beltsville, MD, USA ; Sn, Senegal. Strain New isolates* Group I ISRA 350 ISRA 352 ISRA 354 ISRA 554 Group II Subgroup II.1 ISRA 30 ISRA 319 ISRA 351 ISRA 353 ISRA 355 ISRA 356 ISRA 357 ISRA 361 ISRA 362 ISRA 363 ISRA 364 ISRA 365 ISRA 553 ISRA 555 ISRA 556 ISRA 557 ISRA 558 ISRA 559 ISRA 561 ISRA 562 ISRA 563 ISRA 564 ISRA 566 ISRA 567 ISRA 568 ISRA 569 ISRA 570 ISRA 571 ISRA 572 ISRA 573 ISRA 574 ISRA 575 ISRA 576 ISRA 577 ISRA 578 ISRA 579 ISRA 580
Host plant or origin
Geographical origin
Source or reference
Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris
Bassin arachidier, Sn Sylvopastorale, Sn Sylvopastorale, Sn Sylvopastorale, Sn
This work This work This work This work
Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris
Niayes, Sn Niayes, Sn Bassin arachidier, Sn Sylvopastorale, Sn Bassin arachidier, Sn Bassin arachidier, Sn Sylvopastorale, Sn Bassin arachidier, Sn Sylvopastorale, Sn Bassin arachidier, Sn Bassin arachidier, Sn Bassin arachidier, Sn Bassin arachidier, Sn Bassin arachidier, Sn Sylvopastorale, Sn Sylvopastorale, Sn Sylvopastorale, Sn Fleuve, Sn Fleuve, Sn Fleuve, Sn Fleuve, Sn Bassin arachidier, Sn Fleuve, Sn Bassin arachidier, Sn Fleuve, Sn Fleuve, Sn Fleuve, Sn Fleuve, Sn Bassin arachidier, Sn Bassin arachidier, Sn Fleuve, Sn Fleuve, Sn Fleuve, Sn Fleuve, Sn Fleuve, Sn Fleuve, Sn Gambia
This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work
International Journal of Systematic and Evolutionary Microbiology 50
161
A. Diouf and others Table 1 (cont.) Strain
Host plant or origin
Geographical origin
ISRA 581 ISRA 582 ISRA 583 ISRA 584 ISRA 585 ISRA 586 ISRA 587 ISRA 588 Subgroup II.2 ISRA 21 ISRA 77 ISRA 78 ISRA 560 ISRA 565 Subgroup II.3 ISRA 27 ISRA 59 ISRA 61 ISRA 69 Reference strains Rhizobium tropici Type A CFN 299T
Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris
Gambia Gambia Gambia Gambia Gambia Bassin arachidier, Sn Bassin arachidier, Sn Bassin arachidier, Sn
This work This work This work This work This work This work This work This work
Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris
Casamance, Sn Casamance, Sn Casamance, Sn Fleuve, Sn Fleuve, Sn
This work This work This work This work This work
Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris
Niayes, Sn Niayes, Sn Niayes, Sn Niayes, Sn
This work This work This work This work
Phaseolus vulgaris
Brazil
Martinez-Romero et al. (1991) LMG
Colombia
Martinez-Romero et al. (1991) LMG LMG
Mexico Brazil Mexico Mexico Mexico
Segovia et al. (1993) CIFN CIFN CIFN CIFN Segovia et al. (1993)
LMG 9502 Type B CIAT 899T LMG 9519 LMG 9518 Rhizobium etli CFN 42T BRA 5 F6 F8 F16 Viking 1 USDA 9041 USDA 2667 Rhizobium leguminosarum biovar phaseoli LMG 8820 biovar viciae USDA 2370 biovar trifolii LMG 8819 Rhizobium leguminosarum LMG 9505 LMG 6122 LMG 9504 316C10A
162
Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris
Source or reference
Phaseolus vulgaris
LMG
Pisum sativum
CIFN
Trifolium pratense
LMG
Trifolium repens
Australia
LMG LMG LMG
International Journal of Systematic and Evolutionary Microbiology 50
Phaseolus vulgaris-nodulating rhizobia Table 1 (cont.) Strain Rhizobium galegae LMG 6214T LMG 6215 Rhizobium sp. ORS 240 ORS 1181 ORS 248 Mesorhizobium plurifarium ORS 1001 ORS 1004 ORS 1002 Mesorhizobium loti LMG 6125T Sinorhizobium saheli ORS 609T ORS 600 Sinorhizobium terangae ORS 51 ORS 15 ORS 1009T ORS 1016 LMG 6463 Sinorhizobium meliloti LMG 6133T LMG 6130 Agrobacterium biovar 2 LMG 150T Agrobacterium rubi LMG 156T Agrobacterium biovar 1 LMG 196 Agrobacterium vitis LMG 257
Host plant or origin
Geographical origin
Source or reference
Galega orientalis Galega orientalis
Finland USSR
LMG LMG
Pterocarpus lucens Acacia senegal Pterocarpus lucens
Senegal Senegal Senegal
ORSTOM ORSTOM ORSTOM
Acacia senegal Acacia senegal Acacia senegal
Senegal Senegal Senegal
de Lajudie et al. (1998) de Lajudie et al. (1998) de Lajudie et al. (1998)
Lotus corniculatus
New Zealand
LMG
Sesbania canabina Sesbania pachycarpa
Senegal Senegal
de Lajudie et al. (1994) de Lajudie et al. (1994)
Sesbania rostrata Sesbania sp. Acacia laeta Acacia laeta Sesbania rostrata
Senegal Senegal Senegal Senegal Senegal
de Lajudie et al. (1994) de Lajudie et al. (1994) de Lajudie et al. (1994) de Lajudie et al. (1994) de Lajudie et al. (1994)
Medicago sativa Medicago sativa
Virginia, USA Australia
LMG LMG
Rubus ursinus
USA, 1942
crown gall
USA
Vitis vinifera
Crete, Greece
Kersters et al. (1973)
* Grouping according to ITS-PCR-RFLP.
PCR/RFLP of 16S rRNA genes. A bacterial colony was homogenized vigorously into 100 µl of 0±1 % Tween 20 solution. The cell suspension was heated at 95–100 °C for 10 min and was directly used for PCR assay. DNA was amplified by mixing 3 µl of the suspension with the 16S PCR buffer (10 mM Tris}HCl, pH 8±3 ; 50 mM KCl ; 0±01 % gelatin), 2 µl each of the 10 mM dNTPs (dATP, dCTP, dGTP, dTTP), 1±5 µl 100 mM MgCl , 1 µl each primer fD1 # and rD1 (Weisburg et al., 1991) which correspond to positions 8–27 and 1524–1540 respectively, of the E. coli 16S rRNA gene and 80±2 µl sterilized water. The mixture was sealed with a thin layer of paraffin oil and heated for 3 min at 95 °C. A 2±5 µl aliquot of diluted Taq polymerase in Extender and deionized water (1 : 1 : 3, by vol.) was added to the mixture. The PCR amplification was carried out in a 100 µl reaction volume and performed with a thermal reactor HYBAID. The PCR temperature profile was as follows : initial denaturation at 94 °C for 3 min ; 35 amplification cycles (denaturation 94 °C for 1 min, primer annealing 55 °C for 1 min and extension 72 °C for 2 min) ; final extension at 72 °C for 3 min. International Journal of Systematic and Evolutionary Microbiology 50
An excess of restriction enzymes (5 U}reaction) was used to digest a 10 µl aliquot of PCR product. Restricted DNAs with HhaI, HinfI, MspI and Sau3AI were analysed by horizontal agarose (3 %, w}v) gel electrophoresis carried out at 80 V for 3±5 h. Gels were stained in an aqueous solution of ethidium bromide (1 mg ml−") and photographed under UV illumination using a Polaroid 55 positive}negative film. Auxanographic tests. API galleries (API 50CH, API 50AO
and API 50AA ; bioMe! rieux) were used to test the assimilation of 147 organic compounds as sole carbon sources (Kersters et al., 1984). Inocula were obtained from 36 h YMA slant cultures. After incubation of the galleries for 1, 2, 4 and 7 d at 30 °C, growth of the strains was checked and scored as described previously (Kersters et al., 1984). The levels of interstrain similarity (S) were calculated by using a similarity distance coefficient derived from the Canberra metric coefficient (dCanb) (Sneath & Sokal, 1973) as follows : S ¯ 100¬(1®dCanb). Plant nodulation tests. Phaseolus vulgaris, Vigna unguiculata and Glycine max seeds were surface sterilized successively
163
A. Diouf and others Table 2. Allele profiles at 12 enzyme loci forming 13 ETs among reference strains of R. etli and R. tropici and Rhizobium strains isolated from Phaseolus vulgaris in West Africa ET Strain
Alleles at the following enzyme loci* IDH MDH G6P
1 2 3 4 5 6 7 8 9 10 11 12 13
ISRA 27, ISRA 59 ISRA 61 ISRA 69 Groups II.1 & II.2 strains Group I strains R. etli BRA 5 R. etli CFN 42T R. tropici type A CFN 299T R. tropici type B CIAT 899T R. etli F 6 R. etli F 8 R. etli F 16 R. etli Viking 1
7 7 7 5 5 5 5 5 5 5 7 7 5
5 5 5 5 7 5 5 6 7 5 5 5 5
XDH
4 5 4 5 3 5 5 3 3 4 5 4 3
HEX GD2
6 6 6 5 6±5 6 5 6 6±5 6 6 5±5 6
7 7 7 5 5 5 5 5 5 5 7 7 5
7 7 7 5 5 5 5 5 5 5 7 7 5
ACO 7 7±2 7±2 5 5 5 5 5 5 5 7 7 5
ALA PGM IPO 4 4 4 3 3 3 3 3 3 3 4 4 3
7 7 7 5 5 5 5 5 5 5 7 7 5
5 5 5 5 7 5 5 3 5 5 5 5 5
EST1
EST2
4 4 3±5 3 6 3 4±5 6 6 4 4 3 5±5
5 5 5 5 8 5 5 6±5 8 5 5 5 5±5
* IDH, Isocitrate dehydrogenase ; MDH, malate dehydrogenase ; G6P, glucose-6-phosphate dehydrogenase ; XDH, xanthine dehydrogenase ; HEX, hexokinase ; GD2, glutamate dehydrogenase ; ACO, aconitase ; ALA, alanine dehydrogenase ; PGM, phosphoglucomutase ; IPO, indophenol oxidase ; and EST, esterases. Table 3. Groupings of ISRA Rhizobium strains nodulating Phaseolus vulgaris using PCR-RFLP analysis of the 16S–23S RNA intergenic spacer .....................................................................................................................................................................................................................................
−,
DNA not digested with the corresponding enzyme.
Group or subgroup of strains
Restriction pattern of amplified ITS gene digested with :
Group I Subgroup II.1 Subgroup II.2 Subgroup II.3
II.1 M
1
2
AluI
DdeI
HhaI
HinfI
MspI
PalI
RsaI
TaqI
A1 A2 A2 A4
D1 D2 D2 D4
Hh1 Hh2 Hh2 Hh4
Hi− Hi− Hi3 Hi−
M1 M2 M2 M4
P1 P2 P2 P4
R1 R− R− R−
T1 T2 T2 T4
I 3
4
II.1 5
6
7
8
9
II.3
I.1
10 11 12 13 14 15 16 M
.....................................................................................................
Fig. 1. Restriction patterns of PCR-amplified fragment of 16S–23S rDNA digested with TaqI. Lanes : M, molecular mass marker ; (subgroup II.1) 1, ISRA 351 ; 2, ISRA 355 ; 3, ISRA 553 ; 5, ISRA 556 ; 6, ISRA 558 ; 7, ISRA 562 ; 8, ISRA 563 ; 9, ISRA 567 ; 10, ISRA 570 ; 11, ISRA 576 ; 12, ISRA 586 ; 13, ISRA 77 ; 16, ISRA 319 ; Group I ; 4, ISRA 554 ; (subgroup II.3) 14, ISRA 27 ; 15, ISRA 59.
with 95 % (v}v) ethanol for 3 min and 0±1 % HgCl for 3 min # Acacia followed by several washes in sterile distilled water. senegal, Leucaena leucocephala, Acacia tortilis subsp. raddiana, Faidherbia albida, Sesbania rostrata and Acacia seyal seeds were scarified and surface-sterilized with concen164
trated sulfuric acid for 14, 20, 25, 30, 45 and 60 min, respectively. After treatment, the seeds were washed several times with water to eliminate any trace of acid and HgCl . # The seeds were first allowed to germinate in sterile Petri dishes containing 0±8 % water agar for 24–48 h, then International Journal of Systematic and Evolutionary Microbiology 50
Phaseolus vulgaris-nodulating rhizobia Table 4. Number of alleles and genetic diversity (h) at the loci encoding the 12 enzymes in ETs observed in the Rhizobium strains ISRA isolated from Phaseolus vulgaris and the reference strains .................................................................................................................................................
h ¯ 1®Σxi#(n}(n®1)) where xi is the frequency of the ith allele and n is the number of ETs. Enzyme locus*
Thirteen ETs†
Four ETs‡
No. alleles
h
No. alleles
h
IDH MDH G6P XDH HEX GD2 ACO ALA PGM IPO EST1 EST2
2 3 3 3 2 2 3 2 2 3 5 4
0±513 0±011 0±717 0±616 0±513 0±513 0±590 0±513 0±513 0±295 0±846 0±527
2 1 2 2 2 2 3 2 2 1 3 1
0±499 0±000 0±667 0±499 0±499 0±499 0±832 0±499 0±499 0±000 0±832 0±000
Mean
2±83
0±547
1±91
0±444
* For abbreviations of enzyme loci see the footnote to Table 2. † The 13 ETs of ISRA and reference strains. ‡ The four ETs of ISRA R. etli strains.
transferred to tubes containing Jensen slant agar (Vincent, 1970) for root nodulation tests (6–8 plants were routinely tested with each strain). All 58 new isolates were tested for their ability to nodulate these plants.
RESULTS AND DISCUSSION Collection of strains
Fifty-eight rhizobial isolates (Table 1) were purified from root nodules of common bean and included in the Senegalese Institute of Agricultural Research (ISRA)}Microbiological Resources Center (MIRCEN) culture collection. These isolates originate from different geographical and ecological areas in Senegal and Gambia, including fields in the Niayes zone where common bean is mainly cultivated in Senegal, and were obtained either by direct isolation from naturally occurring nodules or by trapping Rhizobium on sterile young plants inoculated with collected soil samples.
Morphological and physiological characteristics
All isolates were fast growers and, based on their morphological and physiological characteristics, we defined two groups (I and II) among them. In YM liquid medium, strains ISRA 350, 352, 354 and 554 (forming the so-called group I) grew at temperatures up to 44 °C whereas the maximum growth temperature of the 54 other isolates (forming the so-called group II) was 40 °C. On PY medium, the texture of colonies of group I strains was creamy whereas colonies of group II had a gummy texture. Strains of group I were able to grow on LB medium whereas strains of group II could not grow on this medium. These morphological and physiological characteristics of group I strains such as the ability to grow on LB medium and colony morphology on PY medium as well as their maximal growth temperature matched characteristics reported for R. tropici (Martı! nez-Romero et al., 1991). On the other hand, group II strains shared characteristics of R. etli (Segovia et al., 1993).
.....................................................................................................
Fig. 2. Similarity among 13 ETs of ISRA Rhizobium strains isolated from common bean (Phaseolus vulgaris) and reference strains based on electrophoretically detectable allele variation at 12 enzyme loci. *Values in parentheses correspond to the number of strains in each ET. †Only one strain was represented for each ET. International Journal of Systematic and Evolutionary Microbiology 50
165
A. Diouf and others
a separate type with three subgroups, one major (subgroup II.1, 45 strains, see Table 1), and two minor (subgroups II.2 and II.3). The only difference between subgroups II.1 and II.2 was that DNA of the former could not be digested by HinfI. More important were the differences exhibited by the four strains of subgroup II.3 (see Table 1) showing a separate profile. RsaI restricted only ITS amplificates of strains from group I. MLEE
.................................................................................................................................................
Fig. 3. Dendrogram showing the similarity among the electrophoretic protein patterns (SDS-PAGE) of ISRA and reference strains based on the mean correlation coefficient values which were grouped by the unweighted pair group method with averages.
PCR/RFLP of 16S–23S ITS region
Analysis of the PCR product of the 16S–23S ITS DNA region was reported as a useful method to evidence diversity among bacterial populations even at the intraspecific level (Barry et al., 1991 ; Jensen et al., 1993 ; Laguerre et al., 1996). The PCR amplified 16S–23S rDNA intergenic spacer regions of the 58 new isolates were digested with eight restriction enzymes, leading to polymorphic patterns (Table 3, Fig. 1). Four different groups of strains were identified. One corresponded to the four group I strains which shared the same restriction pattern type. Group II strains formed 166
The genetic relationships between the 58 isolates and reference strains of Rhizobium tropici and Rhizobium etli were further examined by performing MLEE, a technique previously employed in taxonomic studies of rhizobia that nodulate bean plants (Pin4 ero et al., 1988 ; Martı! nez-Romero et al., 1991 ; Segovia et al., 1991 ; Souza et al., 1994). The twelve enzyme loci analysed were polymorphic with a number of alleles ranging from two to five electromorphs. The mean number of alleles was 2±83 (Table 4). There were 13 distinctive multilocus genotypes or ETs as indicated in Table 2. The electrophoretic mobilities at the twelve enzyme loci showed five ETs among the 58 isolates. The genetic diversity at the enzyme locus for the four ETs of the 54 ISRA strains of group II was 0±44 (Table 4). On the basis of the analysis of the genetic distance, the rhizobial strains nodulating bean in Senegal and Gambia are clustered into two groups. The four strains of group I (ISRA 350, ISRA 352, ISRA 354 and ISRA 554) corresponded to ET 5 and grouped with R. tropici type B. Group II strains exhibited four ETs, different from, but all similar to, those of R. etli. The majority of group II strains (the 50 strains of subgroup II.1 and II.2) have a single ET 4, highly similar to that of the type strain of R. etli CFN 42T. The four remaining strains of group II (belonging to subgroup II.3) corresponded to ET 1 (ISRA 27 and ISRA 59), ET 2 (ISRA 61) and ET 3 (ISRA 69), all similar to other R. etli reference strains (Table 2, Fig. 2). Both group II and R. etli reference strains showed ET heterogeneity among them but their ETs all clustered together. Nour et al. (1994) reported good agreement of results obtained by RFLP analysis of the rDNA 16S–23S intergenic spacer and MLEE for rhizobia nodulating chickpea (Cicer arietinum). Here both methods confirmed on the whole the distinction of the two main groups I and II among the new isolates, similar to R. etli and R. tropici type B, but with different subgroupings : three ETs were found among subgroup II.3 of ITS-PCR-RFLP, while two ITS-PCR-RFLP subgroups were distinguished among the single ET 4. SDS-PAGE of total bacterial proteins
The SDS-PAGE whole-cell protein patterns of 54 of the rhizobial isolates from Senegal were scanned and numerically analysed, together with those of reference strains available in our database and representing Rhizobium species nodulating bean plants, R. tropici, International Journal of Systematic and Evolutionary Microbiology 50
Phaseolus vulgaris-nodulating rhizobia Group I M
1
2
3
4
Group II 5
6
7
8
9
Group II
10 11 12 13 14 15 16 17 18 .....................................................................................................
Fig. 4. Restriction patterns of PCR-amplified fragment of 16S rRNA genes digested with MspI. Lanes : M, molecular mass marker ; 1, ISRA 350 ; 2, ISRA 352 ; 4, ISRA 354 ; 5, ISRA 554 ; reference strains 3, R. tropici type B CIAT 899 ; 6, R. tropici type A CFN 299 ; 7, ISRA 27 ; 8, ISRA 59 ; 9, ISRA 61 ; 10, ISRA 69 ; 12, ISRA 30 ; 13, ISRA 77 ; 14, ISRA 353 ; 16, ISRA 566 ; 17, ISRA 577 ; 18, ISRA 584 ; reference strain 11, R. etli CFN 42 ; 15, R. leguminosarum USDA 2370. Group I, restriction pattern types of R. tropici type B ; group II, restriction pattern types of R. etli.
R. etli and R. leguminosarum. Bean isolates from Senegal and Gambia essentially grouped in two separate protein gel electrophoretic clusters. The results are presented as a similarity dendrogram in Fig. 3. The four isolates of group I clustered together at a mean correlation coefficient (r) of 94 %. The majority of group II strains formed a second cluster at a mean correlation coefficient of 89 %. Group I and group II were respectively related to reference strains of R. tropici and R. etli at a moderate mean coefficient correlation of 83±5 %. Strain ISRA 27 (belonging to subgroup II.3) grouped separately from the core of other isolates of group II, closer to R. etli. Two other strains of subgroup II.3, ISRA 59 and ISRA 61, grouped together, outside the big groups I and II. The fourth strain of subgroup II.3, ISRA 69, was not included in this study. PCR-RFLP of 16S rRNA genes
PCR-RFLP analysis of 16S rRNA coding gene has been reported to be useful in Rhizobium taxonomy since results obtained are in good agreement with those from sequence analysis of the 16S rRNA coding gene and DNA–DNA hybridization (Laguerre et al., 1993). Four restriction endonucleases HhaI, HinfI, MspI and Sau3AI were used to digest 16S rRNA gene PCR amplificates of a selection of 29 Senegalese isolates, including all strains of group I, subgroup II.2 and II.3 (Table 1), and 16 representative strains of subgroup II.1 (ISRA 30, ISRA 353, ISRA 355, ISRA 580, ISRA 576, ISRA 584, ISRA 555, ISRA 577, ISRA 562, ISRA 566, ISRA 567, ISRA 570, ISRA 571, ISRA 578, ISRA 581, ISRA 584). The restriction patterns generated by HhaI, HinfI and Sau3AI were similar for all isolates, but two distinct restriction patterns were detected with MspI among our isolates (Fig. 4) : the first was exhibited by group I strains and R. tropici CIAT 899T ; the second pattern was exhibited by group II strains and R. etli CFN 42T. R. tropici strains, genomically described by sequence of their 16S rRNA gene, DNA–DNA hybridization and rDNA organization, were separated into two subgroups, namely type A and type B (Martı! nez-Romero et al., International Journal of Systematic and Evolutionary Microbiology 50
.................................................................................................................................................
Fig. 5. Dendrogram showing the differences of auxanographic characteristics among ISRA strains and reference strains, obtained from an unweighted pair group method with averages cluster analysis of Canberra metric similarity coefficients based on 147 auxanographic characteristics.
1991 ; Geniaux et al., 1993). Restriction patterns of R. tropici type A and of R. tropici type B were different, and the pattern of group I rhizobial strains was related to that of the latter. Analysis of auxanographic results
Only the first acquired isolates were tested for assimilation of 147 organic compounds as sole carbon sources, using the API 50 system. Group I was represented by strains ISRA 352, ISRA 354 and group 167
A. Diouf and others
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Fig. 6. Diagram summarizing results of polyphasic characterization of beannodulating rhizobia in Senegal and Gambia.
II was represented by ISRA 555, ISRA 557, ISRA 558, ISRA 564 and ISRA 362. The reproducibility of the test was good. The mean interstrain similarity values for strains tested in duplicate were between 88 and 92 %. Results for representative strains of several Rhizobium species and related groups (including representatives of the Agrobacterium biovars, but not R. etli) were available in the database of our research group and were included in the numerical analysis. The results are shown as a limited dendrogram in Fig. 5. Group I formed a common cluster with R. tropici (similarity coefficient of 89 %). At a similarity coefficient of 86 %, cluster II could be distinguished as a separate cluster. Host specificity
Bean nodulating rhizobia have been reported to have a broad host range (Hernandez-Lucas et al., 1995 ; Geniaux et al., 1993 ; Amarger et al., 1997). The 58 new nodule isolates could all nodulate their original host plant, Phaseolus vulgaris, and Acacia seyal whereas none of them nodulated Glycine max and Sesbania rostrata. Some differences in host ranges were found among the isolates. Only strains of group I were able to effectively nodulate Leucaena leucocephala, similarly to R. tropici (Martı! nez-Romero et al., 1991). The four strains of group I and the four strains of subgroup II.1 (ISRA 27, ISRA 59, ISRA 61 and ISRA 69) nodulated Faidherbia albida and Acacia senegal. By all the criteria tested and results summarized in Fig. 6, group I African isolates were R. tropici type B strains while group II corresponded to R. etli. Population diversity studies of bean nodulating rhizobia from areas where this plant is native and extensively grown have shown a large genetic diversity. This has been observed both for R. tropici from South American soils (Martı! nez-Romero et al., 1991) and for R. etli in Mesoamerica (Pin4 ero et al., 1988 ; Segovia et 168
al., 1991 ; Eardly et al., 1995). For both species a large number of ETs have been obtained with H values (diversity index) of 0±363 for R. tropici type B strains and larger than 0±5 for R. etli. In comparison we report here only one ET for the R. tropici strains in contrast to the 18 ETs for the type B R. tropici strains analysed previously (Martı! nez-Romero et al., 1991). Similarly, only four ETs were obtained for our R. etli strains while a maximum of 70 ETs was reported in R. etli isolated from a limited geographical area in Mexico (Caballero-Mellado & Martı! nez-Romero, 1999). Also indicative of the restricted genetic diversity of the bean rhizobia population in Africa is the large dominance of a single genotype : 50 strains have a single ET. The limited genetic diversity encountered in Senegal and Gambia may be related to the fact that bean is an introduced crop in Africa. It is remarkable that in West, East and South Africa, bean is naturally nodulated by the same species of rhizobia as those nodulating bean at its site of origin. It has been supposed that bean symbiotic rhizobia are cosmopolitan. An alternative hypothesis was suggested by Sessitsch et al. (1997) to explain the presence of R. etli in Austrian soil. They proposed that the rhizobia was co-introduced with bean seeds. The presence of R. etli on Phaseolus vulgaris seeds was demonstrated, and the rhizobia remain viable on the seeds for years (Pe! rezRamı! rez et al., 1998). We may suppose that R. tropici could be also transported on bean seeds harvested from South America where R. tropici is native. The existence of Rhizobium on seeds may explain the presence of R. etli in Africa and the limited genetic diversity encountered may be related to the founder principle, with only a few clones (founders) migrating and surviving at the introduced place. Africa has been proposed to be the site of origin of the Leguminosae family (Raven & Polhill, 1981), and as such a large diversity of legume species and their symbionts exist. If the symbionts of Phaseolus vulgaris International Journal of Systematic and Evolutionary Microbiology 50
Phaseolus vulgaris-nodulating rhizobia
were originally from Africa, we should recover the largest diversity there, but that does not seem to be the case. It seems more probable that bean symbionts coevolved with their host at the site where they diverged and prospered. ACKNOWLEDGEMENTS We thank bioMe! rieux, Montalieu-Vercieu, France, for supplying API galleries. This work was partially supported by UNESCO grant SC}RP}206.575.6. We are grateful to J. Bakhoum, ORSTOM, Senegal, and M. A. Rogel, CIFN} UNAM, Mexico, for technical assistance. M. G. is indebted to the Fund for Scientific Research-Flanders (Belgium), for research and personnel grants.
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